In microscopic analyses of various medicinal preparations, for instance what is referred to as differential counting of white blood cells, it is possible to use automatic scanning microscope systems. One example of such a microscope system is DiffMaster™ Octavia from CellaVision AB, which automatically scans a blood smear which is positioned on the surface of a microscope slide. During scanning, the microscope system must be able to perform, inter alia, controlled positioning movements of the microscope slide in what is below referred to as the x/y plane whose normal is the optical axis of the microscope.
The range, i.e. the size of the scannable area, in x and y is usually slightly smaller than that of the microscope slide, which is about 1 by 3 inches. The resolution of such positioning movements can now be allowed to have a minimum resolution which is tens of a micrometer. The fact that as much as tens of a micrometer can be allowed largely depends on the progress in image analysis and quick jointless assembling of partial images that has been made in recent years. WO 02/084368, A1 discloses, inter alia, a method in scanning microscoping, which solves the problem of scanning a preparation and later returning to found partial objects also with a microscope where the positioning movements have a relatively high minimum resolution even if they are not quite repeatable.
Scanning of the preparation often occurs in a meandering pattern where the x position is changed basically in each movement and where the y position is changed only when the x position has come close to one of the edges of the microscope slide. For the speed of the microscope, the speed of the x positioning is therefore more important than that of the y positioning.
To be able to focus the image of, for instance, a blood cell to a sharp image on an image sensor in a camera, the scanning microscope must also be able to make controlled changes of the mutual distance between the objective of the microscope and the microscope slide. Such changes in distance usually occur exclusively along the optical axis, below referred to as the z axis, and can, depending on the depth of field of the objective used, have to be so small as 0.1 micrometer, i.e. smaller than a whole wavelength of visible light in vacuum. The range in z must be about a millimeter, depending on how plane the microscope slide is and how perpendicularly to the optical axis the positioning in x/y occurs.
It is further desirable that the automatic scanning microscope will quickly have finished its tasks. For quick and reliable scanning of a preparation, it is not enough to use an image sensor with a large number of picture elements, which gives a great surface coverage per image, or an image sensor with a high image frequency. The speed of the system is also dependent on the scanning microscope having a positioning system which can quickly move the preparation to new desired positions, both perpendicular to and along the optical axis. Many of the positioning movements in a DiffMaster™ Octavia system are associated with fine focusing on found cells. 200 cells per preparation at five focusing movements per cell may be involved. The images of the cells are taken by a CCD sensor with a shutter setting in the order of 1 millisecond, where each pixel in the image corresponds to about 0.1 micrometer on the preparation, which means that the preparation must basically be absolutely immovable during exposure. For each 10 milliseconds that the system oscillates after a focusing movement, a waiting time of about 200·5·0.01 s=10 s will be added to the time it otherwise takes for the microscope to analyse the preparation. It is therefore particularly important that the positioning system can make quick z movements of exactly the desired length, with fundamentally no play when changing the direction of movement and with minimum post-oscillations in x, y and z. As discussed above, it matters less, however, where in a range of for instance (±5 μm, ±5 μm, ±0.05 μm) in (x, y, z) the preparation is immovable.
The speed of the scanning microscope is also dependent on whether the microscope, in terms of image analysis, has access to one or more focusing methods which allow it to use few camera images on each fine focusing occasion. Such focusing principles will not be discussed here but are described, for instance, in U.S. Pat. No. 6,341,180, WO 01/11407, A2 and WO 02/39059, A1.
In addition the requirements as to range, resolution, speed and stability of the positioning movements, there are also requirements as to the production and service costs for a commercially useful positioning system.
There are already a number of known ways of implementing x/y/z positioning systems for scanning microscopes. The various ways of implementation differ from each other, among other things, by which storing principles they use. An ideal bearing principle allows a part journalled in bearings to make the desired movements fundamentally without the presence of friction, while at the same time it holds the stored part, without play, along the other dimensions. The various ways of implementation also differ from each other by what actuators and gear mechanisms they use to perform the desired movements. The known ways of implementing x/y/x positioning systems result in different compromises regarding satisfying all the above-mentioned requirements.
In many cases the implementation of the x/y/z positioning system is divided into an x/y part and a z part, i.e. in a part for movements perpendicular to the optical axis and in a part for movements along the optical axis.
A common way of implementing the x/y positioning is to motorise the traditional microscope stage that is to be found in most microscope stands even in the basic design. Repeatability and resolution are in most cases far better than the about 10 micrometers that are mentioned as a desideratum above, which does in fact not add much extra value to the positioning system of a scanning microscope. However, the linear bearings and actuators in the form of stepping motors with so-called ball bearing screws that are used make the microscope stages expensive to manufacture. Owing to their solid structure, the traditional microscope stages have relatively large movable masses and will therefore have relatively slow acceleration as the speed is changed and also relatively long post-oscillations at a great amplitude for tens of milliseconds.
A common way of implementing the z positioning is to motorise the shaft for the manual wheel in the traditional microscope mechanism which allows the objective, and an optional revolver, to be translated in the z direction relative to the stand of the microscope, also referred to as the frame. There are several drawbacks of this way of motorisation. First, microscope frames are relatively expensive. Second, the z mechanisms are in most cases not completely free of play in their gear mechanisms when changing the direction of movement, which results in less predictable real movements in connection with fine focusing, thus requiring unnecessarily many movements. The purchase price of a motorised x/y stage intended for a microscope slide, including a traditional microscope frame with a motorised z mechanism and a control box for the stepping motors, may exceed one hundred thousand Swedish kronor.
Another known way of implementing the z movement is to insert a piece of piezoelectric crystal between the objective and its attachment to the microscope. Since piezoelectric crystals have a thickness which is dependent on the voltage applied across the crystal, the objective will be moved in the z direction in connection with voltage changes. The piezoelectric crystal then constitutes both bearing and actuator. Piezoelectric crystals are very rapid but involve high costs, mainly since they require voltages in the size of kilovolt, separate power units and accurate insulation of these live wires. Piezoelectric z drives usually have a range of some hundred micrometers. A greater range requires larger crystals, higher voltages and cascade connection of a plurality of cooperating crystals. The crystal, its encapsulation and the separate power unit make the solution cost tens of thousands of kronor to buy.
A further known way of implementing the z movement is to use the principle of a common electromagnetic loud-speaker coil and an associated permanent magnet, which is usually called voice coil in positioning contexts. There exist, in relation to their mass, very strong and thus very rapid such combinations of coil and magnet. For instance, this principle has been used for many years for focusing and radial fine adjustment in connection with optical scanning of discs in CD/DVD players. The great limitation in connection with use in microscopes is that the coil and the associated objective cannot be allowed, for reasons of vibrations, to float quite freely in the magnetic field of the magnet, which means that some kind of bearing must be applied, which only allows z movements. Moreover a position sensor is necessary, having a resolution of tenths of micrometers, and a control system to make the objective immovable for a time of exposure of, for instance, milliseconds and, if something bumps against the microscope, to be able to return to approximately the same position. If in addition the bearing introduces friction in the form of a so-called stick-slip phenomenon, the z position of the objective will be difficult to control.
One way of performing small precision movements without requiring bearings is to use what is referred to as flexures—“böjorgan” (bending means) in Swedish. Other English expressions are “flexible beam”, “flexible sheet”, “flexure hinge” and “bending element”. Below use is made of the expression bending means to designate a construction element which, by being elastically deformable, allows changes of the relative positions or the relative directions of other construction elements connected to the bending means. Below the term flexure is used for a construction comprising at least one bending means.
Flexures are used, for instance, in precisely the mounting of a voice coil in a CD/DVD player and in equipment for adjustments with nanometer resolution of optical components in laser experiments. An example of products is the so-called NanoMax-TS™ series of triaxial stages from Melles Griot. Their range is far from enough compared with the need for range in x/y of the automatic scanning microscoping that has been described above. The stages of the NanoMax series are also not intended for quick motorisation.
Instead of solving the entire positioning of x/y/z in the automatic scanning microscopes with a single system, it is possible to use a y system and an x/z system. The y system can be solved with a simple mechanism, which is allowed to be slower and which can be monitored by means of the invention disclosed in WO02084368. The x/z system should then give quick movements with sufficient resolution and a sufficiently great range and still be inexpensive and sufficiently stable for microscoping. The range requirements are approx. a factor 10 times greater for x compared with z, while the resolution in x can be allowed to be approx. a factor 100 coarser than the one in z.
A flexure which provides biaxial, i.e. two-dimensional, positioning by means of two cascade-mounted bending means, i.e. mounted in succession, is disclosed in U.S. Pat. No. 4,635,887. The flexure allows small movements in two dimensions perpendicular to each other and its principles would work excellently as an x/z system for an automatically scanning microscope if the range requirements in x would be as small as those in z and if it would be cheap to provide actuators with the fine resolution that is required in z.